A structured interdomain linker directs self-polymerization of human uromodulin

Uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn’s disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes.Uromodulin (UMOD) is expressed in the thick ascending limb of Henle’s loop as a GPI membrane-anchored precursor that consists of three EGF-like domains, a domain of unknown function (D8C), and a zona pellucida (ZP) module (1, 2) (Fig. 1A, Top). The latter, containing Ig-like domains ZP-N and ZP-C (3–5), is found in other medically important human glycoproteins linked to infertility (egg coat components ZP1–ZP4), nonsyndromic deafness [inner ear α- and β-tectorin (TECTA/B)], Crohn’s disease [glycoprotein 2 (GP2)], and cancer [TGF-β coreceptors betaglycan (BG) and endoglin (ENG)] (6, 7). Upon processing by Ser protease hepsin (8) at a consensus cleavage site (CCS) C-terminal to the ZP module (9), UMOD sheds a C-terminal propeptide (CTP) that contains a polymerization-blocking external hydrophobic patch (EHP), exposing an internal hydrophobic patch (IHP). This event triggers homopolymerization into filaments that are excreted into the urine (4, 10), where UMOD performs a plethora of biological functions, including protection against urinary tract infections, prevention of kidney stones, and activation of innate immunity (1, 2, 11, 12).Open in a separate windowFig. 1.mMBP-fused UMODp forms filaments like native urinary UMOD. (A) Domain organization of urinary UMOD and recombinant constructs mMBP-UMODp and mMBP-UMODp_XR. EGF domains are indicated by roman numerals. EGF IV identified by this study (brown), ZP-N/ZP-C linker (red), IHP (gray), CCS (magenta), CTP (yellow), and 6His-tag (cyan) are shown. Open circles, inverted tripods, and closed circles represent signal peptides, N-glycans, and GPI anchors, respectively. Electron micrographs of filaments of purified urinary UMOD (B), recombinant full-length UMOD from Madin–Darby canine kidney (MDCK) cells (C), purified elastase-digested urinary UMOD (D), and recombinant mMBP-UMODp from HEK293T cells (E). Yellow squiggles in B–E indicate the zigzag arrangement of UMOD repeats, which is most evident in samples lacking the N-terminal EGF I–III/D8C region. (Scale bars, 100 nm.)Although UMOD activity is strictly linked to its supramolecular state (2), the mechanism of ZP module-dependent assembly remains unclear. Mass spectroscopy (MS) analysis of ZP-C disulfide linkages suggests that there are two types of ZP modules with different structures (13). Type II contains 10 conserved Cys (C_1–7,a,b,8) and both homopolymerizes (UMOD, GP2, and TECTA) and heteropolymerizes (ZP1, ZP2, and ZP4), whereas type I (ZP3) includes eight conserved Cys (C_1–8) and only heteropolymerizes with type II (7, 13, 14). However, MS studies of egg coat protein disulfides are contradictory (15), and type II disulfide linkages C₅–C₆, C₇–C_a, and C_b–C₈ are compatible neither with the fold of ZP3 (3) nor with structures of the ZP-C domain of BG, whose ZP module contains 10 Cys (16, 17). At the same time, interpretation of the latter data in relation to polymerization is complicated by the fact that, like ENG, BG remains membrane-associated and does not form filaments (7, 17).To gain insights into the mechanism of ZP module protein assembly, we carried out X-ray crystallographic studies of the complete polymerization region of UMOD. The structure reveals that a rigid interdomain linker is responsible for maintaining UMOD in a polymerization-competent conformation. This rigid linker is conserved in homopolymeric ZP modules, but it is flexible in the structure of ZP2, also presented in this work, which, together with ZP3, forms heteropolymeric egg coat filaments. Furthermore, ZP module proteins that do not make filaments lack such a linker. Because UMOD and ZP2 show conservation of both disulfide pattern and fold, our data reveal that the interdomain linker, rather than a different ZP-C structure, underlies the ability of UMOD to self-assemble. Accordingly, polymerization-competent UMOD forms a dimer via β-sheet extension and hydrophobic interactions, and disruption of this dimer interface completely abolishes filament formation. Our study yields insights into the formation of an essential polymerization intermediate of UMOD and highlights how an interdomain linker can regulate the biological function of a multidomain protein.